question about cell structure and function
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Question 1. MAB1 is a liver protein of unknown function. In the figure below mice have had their MAB1 gene ”knocked out” (KO) making it impossible to produce MAB1 proteins in their liver cells. MAB1 is normally only expressed in liver cells. Wild-type (WT) mice have not had this gene knocked out and express the MAB1 protein. HepG2 is a liver cell line that normally expresses the MAB1 protein. In the figure below the HepG2 cells have been transfected with small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA. The siRNA used was either one directed towards MAB1 mRNAs or towards a control vWF mRNAs.
Examine the figure below
Albumin secretion from liver cells under altered MAB1 level conditions
A,
(upper panel
bottom panel p n
B,
Green fluorescence indicates how much siRNA entered into the cell during the transfection. Red fluorescent indicates how much MAB1 protein is expressed in the cell 1 day after the siRNA transfection
Albumin secretion levels in blood plasma of MAB1 Knock Out (KO) and Wild Type (WT) pups
secretion) (
) (**,
C,
A. Data General outcomes shown:
1) 2)
3) 4)
Scale bars,
p
< 0.001; KO
n
= 25,WT
n
= 30). HepG2 liver cells treated with MAB-siRNA
compared with control cells treated with control vWF siRNA (vWF has no known role in cell
) (**,
< 0.001;
= 10).
immunofluorescent staining of MAB1 in HepG2 liver cells transfected with MAB1-siRNA or
vWF-siRNA-control.
Western blotting of HepG2 cells showing MAB1 protein level after the cells were treated with
MAB1-siRNA or treated with control vWF-siRNA.
20 μm. Tubulin is the loading control.
Explain what the data in section A above shows about MAB1 for ….a) WT vs. KO
Explain what the data in section A above shows about MAB1 for… b) vWF-siRNA treated
vs. MAB1 siRNA treated
What is siRNA? How does it perform its function in a cell? Why was it used in section B in
the above figure? What were the researchers trying to show?
Why was this western blot in section C done by the researchers? Why is it an important piece
of supporting evidence to the experiments done in parts (A) lower panel.
5) Collectively, what does all the data in the figure suggest about MAB1’s functional role in liver cells.
B. MAB-1 potential functions:
- If MAB-1 is a COPII coat adaptor protein such as sec23 or sec24 that use the SAR-1-GTP interactions (see Chapter 13) how would that explain the data in part A (upper panel) of the figure above.
- If MAB-1 was a vesicular lectin found on the surface of trans-Golgi-derived exocytotic vesicles. And these lectins typically target Phosphatidylinositol 4,5 –PI (4,5) P2 membrane lipids (see chapter 13) how would that explain the data in part A (lower panel-siRNA treated) of the figure above.
- If MAB-1 is an ER resident albumin cargo receptor in the ER membrane (see chapter 13) with its own KDEL sequence and we mutated the KDEL sequence, what would be the short and long-term effects on plasma levels of secreted albumin in such a mutant animal.
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